Telomeres are repeated hexanucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks; furthermore, the asymmetric replication of DNA would lead inevitably to a loss of genetic material, and telomerase, an enzymatic complex that adds telomeric sequences at mitosis, functions to maintain genomic integrity. Telomerase deficiency manifests with short telomeres and loss of both enzymatic activity: its consequences can be measured in vitro and in vivo. Mutations in DKC1 and in TERC (the RNA template subunit of the complex) are etiologic in some cases of dyskeratosis congenita, a constitutional form of aplastic anemia. Mutations in TERT (encoding telomerase, the rate limiting enzymatic component of the complex) occur in apparently acquired aplastic anemia and other diseases. Heterozygous mutations in TERT lead to defective telomere repair and short telomeres due to a mechanism of haploinsufficiency. Male hormones, long used to treat aplastic anemia, act by up regulating TERT transcription and telomerase activity, including in lymphocytes and hematopoietic progenitor cells. While critical telomere shortening often leads to either cell senescence or apoptosis, occasional cells become anneuploid due to end-to-end fusion of chromosomes. Thus, telomere attrition is a mechanism for onco genesis. Telomere length of leukocyte is now measured routinely in our CLIA laboratory by gene amplification using robotic methodology provided by a Quiagen Quiagility and Rotor GeneQ; high throughput analysis is useful both for research and in the clinic, and our procedure is certified for patient data. Measurement of clinical samples is required for the adequate diagnosis of aplastic anemia and is predictive of late events after treatment with immunosuppression, and probably in other clinical circumstances. We now have established singular telomere length analysis SIS (STELA), which relies on amplification using chromosome specific sub-telomeric DNA sequence to detect critical telomere shortening in individual chromosomes. Our clinic continues to see patients with suspected or known telomeropathy, and we are cataloging clinical manifestations and outcomes. We have performed the first direct comparison of two theories of cancer, one based on the acquisition of mutations in tumor suppressor, oncogenes, and genes and other pathways related to cell differentiation, apoptosis, and self renewal., and the other on instability at the chromosome level. We have serial samples from patients with aplastic anemia who evolved to malignant clonal evolution to myelodysplasia (MDS) and acute myeloid leukemia (AML), from which we have analyzed bone marrow myeloid cells for acquired mutations, using CD3 cells from peripheral blood as germ line controls. We examined in late specimens, obtained at the time of evolution to MDS/AML and always associated with monosomy 7, bone marrow from 13 patients. by whole genome sequencing the sequence of almost 125 candidate genes, previously described in MDS and AML. At the same time, we also examined by both q-PCR and STELA telomere attrition in these samples over time. Clinical controls were samples from patients whose aplastic anemia was stable. We found a paucity of genetic events in the group of 13 patients; in two cases we found heterozygous mutations in DNMT3A and in one of these patients additional mutations in several other candidate genes, all present at the time of aplastic anemia and stable through a long course of response to immunosuppression, relapse, and evolution. In contrast, in the remaining 11 patients, no mutations were present in candidate genes. In contrast, patients who underwent clonal evolution showed markedly exaggerated telomere attrition, almost ten times higher rate than in normal individuals or in patients with aplastic anemia with stable disease. STELA also revealed the acquisition of short fragments, especially with XpYp probes, that occurred early and preceded the evolution events. Telomere attrition was also exaggerated in vitro on tissue culture of bone marrow from the same patients over a two week period, likely reflecting selection of cells. These data suggest that chromosome aberrations rather than the acquisition of gene mutations drive the early events in the development of immune-mediated aplastic anemia to monsomy 7, MDS, and AML. As such, the results have implications for mechanisms underlying the relationship between inflammation and carcinoma, as occurs in autoimmune diseases, chronic viral infection, and in immune processes like graft-versus-host disease. In the clinic, we now have identified 136 patients with telomeropathies, based on either extremely low age-adjusted telomere content and/or the identification of mutations in telomerase repair complex genes and shelterin components. TERT mutations were more common than those in TERC. Because of referral bias, most of our patients have hematologic manifestations, but these range widely from typical severe aplastic anemia to chronic aplastic anemia, macrocytic anemic, thrombocytopenia, large granular lymphocytosis, and other unexpected syndromes, such as autoimmune cytopenias and Waldenstrom macrogloubinemia. An individual patient history of liver and lung problems or a family history of hepatic steatosis, cirrhosis, or other abnormalities and pulmonary fibrosis are common but not invariable. A large proportion of patients do not have a suggestive pedigree. Neither physical anomalies nor a family history of multiple malignancies was prominent in the cohort. Patients with mutations and with short telomeres do appear to respond to immunosuppression at approximately the same rate as those with normal telomeres and without mutations, but they may have less robust responses and relapse more frequently. Historically, male hormones have been reported to be effective in some patients with bone marrow failure. Our laboratory studies established that sex hormones increase TERT transcription, a possible mechanism of action in aplastic anemia. Our current prospective clinical trial in patients with telomeropathy employs danazol, a male hormone with minimal virilizing effects. A total of 24 patients have enrolled on protocol. Toxicity has been manageable and most patients do not describe major side effects. There is a high rate of hematologic response: 22 patients are evaluable at six months, 12 were responders, 4 were non-responders and 6 no longer were receiving the study drug. At 12 months, 10/19 evaluable patients were responders, one was non-responder and 8 were no longer on study. Pulmonary fibrosis has not resolved but has remained stable in some cases. Preliminary data suggests telomere length attrition has been minimal with treatments, and in some patients, telomere length appears to have elongated. Full results will be analyzed when all 24 patients have reached their two year time point. In mouse experiments, we were not able to confirm our hypothesis that Peromyscus in the wild had short telomeres compared to bred animals, either obtained through the Chicago Zoo or from a South Carolina breeding facility. Experiments underway examine the influence of mail hormones in our Peromyscus colony. We are also attempting to replicate experiments showing that male hormones, under circumstances of hematopoietic stress, such as bone marrow transplant, chronic radiation, can abrogate telomere attrition. A limitation in these experiments has been variability among animals and also difficulties in applying gene amplification to telomere length measurement in mice with extremely long telomeres compared to humans.